Selenoenzymes use the rare amino acid selenocysteine, the so-called 21st amino acid in the genetic code. Insertion of selenocysteine (Sec) into a protein is much more complicated than the other 20 amino acids because a UGA stop codon must be recoded as a sense codon for Sec and this process requires complex cellular machinery. Any explanation that accounts for the use of Sec in an enzyme must explain why it is needed relative to the use of the more commonly used cysteine (Cys) residue in order to justify maintaining the energetically costly Sec-insertion machinery. The most frequently given reasons for the use of Sec is that it is a type of super-Cys residue that can speed reactions due to selenium's superior chemical reactivity relative to sulfur. If this were true, then we might expect to find the use of Sec widely spread throughout nature instead of its observed rarity. We are pursuing a new hypothesis that explains the biological pressure to maintain the UGA recoding apparatus for Sec. This biological pressure is based upon the superior chemical property of selenium (relative to sulfur) to confer resistance to oxidation and we thus name it the chemico-biological rationale for the presence of Sec in enzymes. Sec can resist oxidation in two ways that Cys cannot. First when Sec is oxidized to seleninic acid (Sec-SeO2-) it can be converted back to the parent form (Sec-SeH) with relative ease compared to the extreme difficulty that the oxidized form of Cys (Cys-SO2-) can be converted to its parent form (Cys-SH). Second, it is much more difficult for Sec-SeO2- to be further oxidized to Sec-SeO3-, while Cys-SO2- can be oxidized to Cys-SO3- relatively easily. We believe both of these facts are unrecognized in the biochemical literature and our experiments will address the hypothesis that Sec only occurs in an enzyme when the enzyme needs to be very resistant to inactivation by oxidation. In other words Sec will substitute for Cys in an enzyme when this Cys-enzyme would otherwise be inactivated due to oxidation of its active-site Cys residue to sulfinic acid (Cys-SO2-). This major hypothesis will be addressed in this study by showing how the selenoenzymes thioredoxin reductase and methionine sulfoxide reductase resist oxidation using both in vitro and in vivo experiments. In the case of thioredoxin reductase we will show how the modular design of the enzyme is such that it carries within itself its own internal rescue system for reducing the Sec- SeO2- residue back to Sec-SeH. Thioredoxin reductase is a major therapeutic target for anti-cancer drugs due to its role in enhancing cell proliferation and regulating cellular apoptotic pathways. Oxidation of methionine to methionine sulfoxide is suspected to play a major role in neurodegenerative disorders such as Parkinson's and Alzheimer's diseases, and understanding how methionine sulfoxide reductase may become inactivated due to oxidation is critical to understanding how antioxidant therapies can best be used to prevent inactivation of the enzyme. The successful completion of the goals of this proposal will provide a universal chemical basis for the nutritional requirement of selenium in humans and other organisms. PUBLIC HEALTH RELEVANCE: Selenium is an essential trace element because it is required for incorporation into a specialized set of enzymes - selenoenzymes that use the rare amino acid selenocysteine. The primary selenoenzyme in this study, thioredoxin reductase is a major cancer target due to its role in preventing apoptosis (which cancer cells must avoid) and promoting cell proliferation. Cancer cells must divide rapidly to cause pathogenesis and require increased expression of thioredoxin reductase to survive.